P2 receptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors (usually designated P2Y or P2Y.sub.n, where "n" is a subscript integer indicating subtype) are believed to differ from ionotropic receptors (usually designated P2X or P2X.sub.n) in that they are based on a different fundamental means of transmembrane signal transduction: P2Y receptors operate through a G protein-coupled system, while P2X receptors are ligand-gated ion channels. The ligand for these P2X receptors is ATP, and/or other natural nucleotides, for example, ADP, UTP, UDP, or synthetic nucleotides, for example 2-methylthioATP.
At least seven P2X receptors, and the cDNA sequences encoding them, have been identified to date. P2X.sub.1 cDNA was cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994) Nature 371:516-519) and P2X.sub.2 cDNA was cloned from PC12 cells (Brake et al. (1994) Nature 371:519-523). Five other P2X receptors have been found in cDNA libraries by virtue of their sequence similarity to P2X.sub.1 and P2X.sub.2 (P2X.sub.3 : Lewis et al. (1995) Nature 377:432-435, Chen et al. (1995) Nature 377:428-431; P2X.sub.4 : Buell et al. (1996) EMBO J. 15:55-62, Seguela et al. (1996) J. Neurosci. 16:448-455, Bo et al. (1995) FEBS Lett. 375:129-133, Soto et al. (1996) Proc. Natl. Acad. Sci. USA 93:3684-3688, Wang et al. (1996) Biochem. Biophys. Res. Commun.220:196-202; P2X.sub.5 : Collo et al. (1996) J. Neurosci. 16:2495-2507, Garcia-Guzman et al. (1996) FEBS Lett. 388:123-127; P2X.sub.6 : Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223:456-460; P2X.sub.7 : Surprenant et al. (1996) Science 272:735-738). For a comparison of the amino acid sequences of rat P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221-2228.
Native P2X receptors form rapidly activated, nonselective cationic channels that are activated by ATP. Rat P2X.sub.1 and rat P2X.sub.2 have equal permeability to Na.sup.+ and K.sup.+ but significantly less to Cs.sup.+. The channels formed by the P2X receptors generally have high Ca.sup.2+ permeability (P.sub.Ca /P.sub.Na.apprxeq.4). The cloned rat P2X.sub.1, P2X.sub.2 and P2X.sub.4 receptors exhibit the same permeability for Ca.sup.2+ observed with native receptors. However, the mechanism by which P2X receptors form an ionic pore or bind ATP is not known.
A variety of tissues and cell types, including epithelial, immune, muscle and neuronal, express at least one form of P2X receptor. The widespread distribution of P2X.sub.4 receptors in the rat central nervous system suggests a role for P2X.sub.4 -mediated events in the central nervous system. However, study of the role of individual P2X receptors is hampered by the lack of receptor subtype-specific agonists and antagonists. For example, one agonist useful for studying ATP-gated channels is .alpha.,.beta.-methylene-ATP (.alpha.,.beta.meATP). However, the P2X receptors display differential sensitivity to the agonist with P2X.sub.1 and P2X.sub.2 being .alpha.,.beta.meATP-sensitive and insensitive, respectively. Furthermore, binding of .alpha.,.beta.meATP to P2X receptors does not always result in channel opening. The predominant forms of P2X receptors in the rat brain, P2X.sub.4 and P2X.sub.6 receptors, cannot be blocked by suramin or PPADS. These two forms of the P2X receptor are also not activated by .alpha.,.beta.meATP and are, thus, intractable to study with currently available pharmacological tools.
A therapeutic role for P2 receptors has been suggested, for example, for cystic fibrosis (Boucher et al. (1995) in: Belardinelli et al. (eds) Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer Acad., Norwell Mass.) pp 525-532), diabetes (Loubatieres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp 337-345), immune and inflammatory diseases (Di Virgilio et al. (1995) in: Belardinelli et al. (eds), supra, pp 329-335), cancer (Rapaport (1993) Drug Dev. Res. 28:428-431), constipation and diarrhea (Milner et al. (1994) in: Kamm et al. (eds.) Constipation and Related Disorders: Pathophysiology and Management in Adults and Children (Wrightson Biomedical, Bristol) pp 41-49), behavioral disorders such as epilepsy, depression and aging-associated degenerative diseases (Williams (1993) Drug. Dev. Res. 28:438-444), contraception and sterility (Foresta et al. (1992) J. Biol. Chem. 257:19443-19447), and wound healing (Wang et al. (1990) Biochim. Biophys. Res. Commun. 166:251-258).
Accordingly, there is a need in the art for specific agonists and antagonists for each P2X receptor subtype and, in particular, agents that will be effective in vivo, as well as for methods for identifying P2X receptor-specific agonist and antagonist compounds. P2 purinoreceptors have been generally categorized as either metabotropic nucleotide receptors or ionotropic receptors for extracellular nucleotides. Metabotropic nucleotide receptors, designated P2Y.sub.n, and the ionotropic receptors, designated P2X.sub.n, are distinguished on the basis of their respective transmembrane signal transduction mechanisms as well as structural differences; the P2Y.sub.n receptors operate through a G protein-coupled system, while the P2X.sub.n receptors are ligand-gated ion channels. The ligand for these receptors may be ATP and/or another natural nucleotide such as ADP, UTP and UDP, or a synthetic nucleotides such as 2-methylthioATP.
At least seven P2X receptors, and the cDNA sequences therefore, have been identified to date. P2X.sub.1 cDNA has been cloned from the smooth muscle of the rat vas deferens (Valera et al. (1994) Nature 371:516-519) and P2X.sub.2 cDNA was cloned from PC12 cells (Brake et al. (1994) Nature 371:519-523). Five other P2X receptors have been found in rat neuronal cDNA libraries by virtue of their sequence similarity to P2X.sub.1 and P2X.sub.2 (P2X.sub.3 : Lewis et al. (1995) Nature 377:432-435, Chen et al. (1995) Nature 377:428-431; P2X.sub.4 : Buell et al. (1996) EMBO J. 15:55-62, Seguela et al. (1996) J. Neurosci. 16:448-455, Bo et al. (1995) FEBS Lett. 375:129-133, Soto et al. (1996) Proc. Natl. Acad. Sci. USA 93:3684-3688, Wang et al. (1996) Biochem. Biophys. Res. Commun.220:196-202; P2X.sub.5 : Collo et al. (1996) J. Neurosci.16:2495-2507, Garcia-Guzman et al. (1996) FEBS Lett. 388:123-127; P2X.sub.4 : Collo et al. (1996), supra, Soto et al. (1996) Biochem. Biophys. Res. Commun. 223:456-460; P2X.sub.7 Surprenant et al. (1996) Science 272:735-738. For a comparison of the amino acid sequences of rat P2X receptors see Buell et al. (1996) Eur. J. Neurosci. 8:2221-2228. -38-
Native P2X receptors form rapidly-activated, nonselective cationic channels that are activated by ATP. P2X.sub.1 and P2X.sub.2 have equal permeability to Na.sup.+ and K.sup.+ but significantly less to Cs.sup.+. The channels formed by the P2X receptors generally have high Ca.sup.2+ permeability (P.sub.Ca /P.sub.Na.sup.4). The cloned rat P2X.sub.1, P2X.sub.2, and P2X.sub.4 receptors exhibit the same permeability for Ca.sup.2+ observed with native receptors. However, the mechanism by which P2X receptors form an ionic pore or bind ATP is not known.
A variety of tissues and cell types, including epithelial, immune, muscle and neuronal, express at least one form of P2X receptor. (As there appear to be heteromeric as well as homomeric P2X receptors in certain tissues, and without intending to be bound by theory, it is believed that some cells in fact express two or more receptor forms.) Moreover, the association of particular receptor types with certain tissues suggests a functional specialization for some of these receptors. For example, the widespread distribution of P2X.sub.4 receptors in the rat central nervous system suggests a role for P2X.sub.4 -mediated events in the central nervous system.
Unfortunately, study of the role of individual P2X receptors is hampered by the lack of receptor subtype-specific agonists and antagonists. One agonist useful for studying ATP-gated channels is, -methylene-ATP (, meATP); however, the P2X receptors display differential sensitivity to the agonist with P2X.sub.1 and P2X.sub.2 being, meATP-sensitive and insensitive, respectively. Furthermore, binding of, meATP to P2X receptors does not always result in channel opening. The predominant forms of P2X receptors in the rat brain, P2X.sub.4 and P2X.sub.6 receptors, cannot be blocked by suramin or pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid ("PPADS"). These two forms of the P2X receptor are also not activated by, meATP and are, thus, intractable to study with currently available pharmacological tools.
Similarly, a variety of P2Y receptors have been identified and cloned from tissues such as erythroleukemia cells (P2Y.sub.1), airway epithelium (P2Y.sub.2 and P2Y.sub.6), and placenta (P2Y.sub.4).
A potential therapeutic role for P2 purinoreceptors has been suggested, e.g., for cystic fibrosis (Boucher et al. (1995) in: Belardinelli et al. (eds.) Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology (Kluwer Acad., Norwell Mass.) pp 525-532), diabetes (Loubatieres-Mariani et al. (1995) in: Belardinelli et al. (eds), supra, pp 337-345, immune and inflammatory diseases (Di Virgilio et al. (1995) in: Belardinelli et al. (eds), supra, pp 329-335), cancer (Rapaport (1993) Drug Dev. Res. 28:428-431), constipation and diarrhea (Milner et al. (1994) in: Kamm et al. (eds.) Constipation and Related Disorders: Pathophysiology and Management in Adults and Children (Wrightson Biomedical, Bristol) pp 41-49), behavioral disorders such as epilepsy, depression and aging-associated degenerative diseases (Williams (1993) Drug. Dev. Res. 28:438-444), contraception and sterility (Foresta et al. (1992) J. Biol. Chem. 257:19443-19447 ) and wound healing (Wang et al. (1990) Biochim. Biophys. Res. Commun. 166:251-258). There additionally may be possibilities in the treatment of pain, particularly in connection with P2X.sub.3 homomeric receptors and P2X.sub.2 /X.sub.3 heteromeric receptors.
Accordingly, for both research and therapeutic purposes there is a need in the art for specific agonists and antagonists for each purinoreceptor subtype and, in particular, agents that will be effective in vivo, as well as for methods for identifying purinoreceptor-specific agonist and antagonist compounds.